Stabilization of viral vaccine and inactivation of adventitious tissue culture contaminants therein



United States Patent STABILIZATION OF VIRAL VACCINE AND IN- ACTIVATION0F ADVENTITIOUS TISSUE CUL- TURE CONTAMINANTS TI-EREKN Joseph L. Melnickand Craig Wallis, Houston, Tex., as-

signors to Baylor Medical Foundation, Houston, Tex., a non-profitcorporation of Texas No Drawing. Filed Get. 5, 1961, Ser. No. 143,038

2 Claims. (Cl. 16778) This invention relates to certain viruses of theribonucleic acid-containing (RNA) type and to vaccines made from them,particularly to live poliovirus vaccines. Moreover, it relates tomethods for stabilizing certain RNA viruses and vaccines made from themand to methods for separating extraneous viruses inadvertentlyincorporated into the virus harvests from which the vaccine is prepared.The present application is a continuation-inpart of our applicationSerial No. 128,953, filed August 3, 1961, now abandoned. Since theintroduction of tissue culture procedures attenuated strains of manyviruses have been cultivated. These strains have lost their ability tocause clinical symptoms but retain the ability to multiply and when usedin a vaccine to protect the host from subsequent infection with virulentstrains of the same virus. The infectivity of the virus is essential tothese actions and therefore to the efficacy of the live attenuated virusvaccine.

The problem of retaining the infectivity of the virus prior to use iswell recognized. Generally, special refrigeration requirements must bemet if the vaccine products are to have even a short usable life period.The expenses of refrigeration adds materially to their price. But addingeven more materially to the cost of the vaccines is the fact thatbecause of the short usable life period when held at above freezingtemperatures much of the vaccine may be returned to the manufacturerunused.

Prior art procedures for retaining infectivity of live virus vaccinesnotably vaccinia virus include in addition to storage in the frozenform, storage in the dried form, cold storage in combination withglycerol, and addition of sodium chloride, potassium chloride, orammonium chloride in concentrated solutions. These methods except forstorage under refrigeration which has only a limited usefulness, whilesuitable for viruses of the DNA- type such as vaccinia virus, are lesssuitable for the RNA- type viruses which tend to be less stable thanvaccinia virus. The polioviruses in the suspensions utilized for oralvaccines are particularly sensitive to deterioration, especially whendried, or even in the fluid state when held at temperatures abovefreezing, when contrasted to the crude tissue extracts of infectedtissue utilized for smallpox vaccine.

Another problem arising in the preparation of virus tissue culturevaccines is the inadvertent incorporation of viruses present asspontaneous contaminants of the cell cultures into the virus harvestfrom which the vaccine is prepared. In the case of live poliovirusvaccines, the virus for which is usually grown in monkey kidneycultures, the contaminating virus may be carried along with the vaccinevirus and find its way into the final vaccine. Thus many lots of livepoliovirus vaccine, particularly those prepared from rhesus monkey(Macaca mulatta) kidney cultures, have contained relatively highconcentrations of vacuolating virus (as high as 10 TCD per ml. ofvaccine).

It is an object of our invention to provide an inexpensive and simplemethod for increasing the period of time during which live poliovirusvaccines will retain their full viability and infectivity even attemperatures up to 50 C. and to provide live poliovirus vaccines whichare usable for several months or more when refrigerated at above "Icefreezing temperatures customarily used for biological productsgenerally, and for weeks when kept at room temperature. It is a furtherobject of our invention to provide a means of eliminating extraneousviruses present in a harvest for vaccine production while stabilizingthe desired virus for use in the vaccine.

We have made the surprising discovery that divalent metal cations suchas those of magnesium, calcium, zinc, cobalt, or manganese in highconcentrations will delay the deterioration of types 1, 2, and 3,attenuated and virulent polio strains at temperatures from 0 C. to morethan 50 C., while not altering the d and t (rot/40 C.) markers or thelow monkey neurovirulence properties characterizing the attenuatedstrains. Furthermore, while the presence of these divalent metal cationsstabilized the particularly sensitive RNA poliovirus, it had nostabilizing effect on the extraneous viruses present as contaminants inthe virus suspensions obtained from monkey kidney cultures, thuspermitting inactivation of the latter. Surprisingly, we also discoveredthat the stabilization of the RNA virus effected by the divalent cationwas independent of the anion of the salt added.

In this aspect our invention is carried out'by adding concentratedsolutions containing divalent metal cations, preferably magnesium in theform of magnesium chloride, to suspensions of the desired live virusuntil the final concentration is from approximately 0.5 M to saturatedsolutions. The resulting virus suspension may be used as an oral vaccinein this form, with or without other additives, preservatives, extendersor the like or it may be treated to inactivate extraneous virusespresent by heating to 50 C. t

for one to two hours and then used with or without the aforementionedadditives, etc.

In another aspect of our invention we have made the surprising discoverythat non-toxic trivalent cations, for example, trivalent aluminum, willalso protect types 1, 2 and 3 attenuated poliovirus from thermalinactivation without altering their viability and infectivity. Trivalentaluminum is used preferably in the form of'aluminum chloride or aluminumsulfate with the final concentrations being from about 0.0001 molar to0.1 molar.

In this aspect of our invention, for example, when aluminum chloride isadded to oral polio vaccine strains so that the trivalent aluminumcation concentration is between 0.0001 M and 0.1 M, the suspension maybe heated to 48 C. for one hour with no loss in infectivity. At theseconcentrations, the trivalent salt enhances the thermal deterioration ofviruses sometimes present as contaminants of tissue culture cells inwhich the poliovirus vaccine strain is grown. This aspect of ourinvention also permits the easy destruction of the undesirablecontaminating virus in the poliovirus suspension.

For example, aluminum chloride added to result in the concentrations setforth to attenuated poliovirus suspensions prepared in monkey kidneycultures contaminated with vacuolating SV virus and the resultingsuspension heated for one hour at 48 C. destroyed the vacuolating SVvirus leaving the desired poliovirus behind intact and fully infectious.

In another aspect of our invention we have found that mixtures of thedivalent metal cations and non-toxic trivalent metal cations when addedto suspensions of the live virus in amounts sufficient to form theconcentrations set forth result in stabilized poliovirus suspensionswhich are free from viable extraneous nonpolioviruses. To eliminate theinfectious extraneous viruses, the suspensions containing the mixturesof divalent and trivalent cations may stand at any temperature up to 50C. for periods of time longer than one hour and be used as an oralvaccine in this form with or without other additives, preservatives,extenders and the like.

The following examples are given to illustrate our inn; it;

vention and are not to be regarded as limitations of their invention,may variations of which are possible without departing from its spiritor scope.

Example I Example [I The vaccine of Example I was heated to 50 C. andmaintained at this temperature for 1 hour which inactivated theinfective vacuolating SV virus present as a contaminant.

Example Ill As in Example I, a 2 molar sterile solution of magnesiumchloride was added to live poliovirus vaccine type 2. This solution wasalso added to live poliovirus vaccine type 3 and to a mixture ofpoliovirus vaccine types 1, 2 and 3. These resulted in stabilized virussuspensions usable as vaccines containing magnesium chloride in 1 molarconcentration.

Example IV The stabilized virus suspensions, the vaccines, of ExampleIII were treated as in Example II which inactivated the infectivevacuolating virus.

Example V In this example, a 1 molar sterile solution of magnesiumchloride prepared as in Example I was added to an equal volume of thepoliovirus vaccines of Examples I and II. This resulted in stabilizedvirus suspensions usable as vaccines, containing magnesium chloride in0.5 molar concentration.

Example VI The stabilized virus suspensions, the vaccines, of Example VWere heated as in Example II which inactivated the infective vacuolatingSV virus present as a contaminant.

Example VIl In this example, 1 part of the vaccines of Examples I andIII were diluted with parts of a saturated sterile solution of magnesiumchloride prepared as in Example I. This resulted in stabilized virussuspensions usable as vaccines, containing magnesium chloride in 10/11saturation.

Example VIII The stabilized virus suspensions, the vaccines of ExampleVII were treated as in Example II which inactivated the infectivevacuolating SV virus present as a contaminant.

Example IX In this example, sterile solutions of magnesium sulfate,calcium chloride, zinc chloride, manganese sulfate and cobalt chloridewere made in accordance with and in the concentrations set forth andadded to the poliovirus vaccines set forth in Examples I, III, V andVII. These resulted in stabilized virus suspensions usable as vaccinesin the concentrations indicated.

Example X The stabilized virus suspensions, the vaccines, of Example IXwere treated as in Example II Which inactivated the infectivevacuolating virus present as a contaminant.

The volumes of poliovirus vaccine and stabilizing salt solution can bevaried as desired as long as the final salt concentration is above 0.4molar.

It is significant that by heating the stabilized virus suspension, thevaccine, that the infective vacuolating virus are inactivated sinceheating of the poliovirus vaccine without the divalent metal cation doesnot inactivate the infective vacuolating virus. The poliovirussuspension may be heated from 0 C. up to 50 C. or not heated at all, asdesired. By heating the poliovirus suspension to 50 C. for at least onehour, the inlfective vacuolating virus is inactivated as previouslymentioned. Preferably, at high temperatures, such as 50 C., the heatingshould not exceed two hours as prolonged heating at such temperaturescauses a loss of effectiveness of the vaccine suspension. Also, theheating at 50 C. can be from about five minutes to one hour as asubstantial amount of the infective vacuolating virus is inactivated infive minutes. Heating at 50 C. for about one hour is preferred since itinsures inactivation of the inifective vacuolating virus present as acontaminant without destroying the effectiveness of the stabilized virussuspension.

Example XI Aluminum chloride was dissolved in distilled water andfiltered to make a sterile 0.2 M solution. An equal volume of type Iattenuated vaccine strain of poliovirus was mixed with an equal volumeof this solution, which resulted in a stabilized virus suspension,usable as a vaccine, containing aluminum chloride at 0.1 molarconcentration.

Example XII The vaccine of Example XI was heated to 48 C. and maintainedat this temperature for one hour which inactivated the infectivevacuolating virus present as a contaminant.

Example XIII As in Example XI, an 0.2 molar sterile solution of aluminumchloride was added to live poliovirus vaccine type 2 and also to livepoliovirus vaccine type 3 and to a mixture of poliovirus vaccine types1, 2 and 3. These resulted in stabilized virus suspensions usable asvaccines containing aluminum chloride in 0.1 molar concentration.

Example XIV The stabilized virus suspensions, the vaccines, of ExampleXIII were treated as Example XII which inactivated the infectivevacuolating virus present as a contaminant.

Example XV In this example, aluminum chloride in a concentration of0.0002 M and also in a concentration of 0.2 M was added to livepoliovirus vaccines as described in Examples XI to XIV, inclusive. Theseresulted in stabilized virus suspensions usable as vaccines containingaluminum chloride in 0.0001 M and 0.1 M concentrations, usable asvaccines and when heated as set forth, freed the vaccines of infectivevacuolating SV virus. At concentrations of 0.04 molar or greater,vacuolating 8V virus was inactivated by allowing the virus suspension toremain at temperatures below 48 C.

Example XVI In this example, aluminum sulfate was substituted foraluminum chloride in Examples XI through XV in the concentrations setforth. These resulted in stabilized virus suspensions usable as vaccinesin the same concentrations indicated and, when heated as described,inactivated the infective vacuolating VlIIllS.

Example XVII In this example a mixture of the divalent and trivalentcations of the preceding examples in the concentrations set forth wereadded in equal proportions to live poliovirus vaccines as set forth inthe preceding examples and in accordance therewith. This resulted in astabilized virus suspensions usable as Vaccines in the concentrations offrom 0.0001 to 0.1 molar aluminum and of from 0.4 molar to saturation ofmagnesium, calcium, zinc, manganese and cobalt. Also, the infectivevacuolating virus was inactivated when the mixtures of virus and saltswere allowed to stand at temperatures of 50 C. or below, for example, at25 C. for one hour.

Thus, trivalent cations can also be used, either alone or combined withthe divalent cations, to provide stabilized vaccines tree of undesirablecontaminating viruses.

While for the purpose of disclosure, the specification has been directedprimarily toward the stabilization of polioviruses and vaccines madefrom them, other of the RNA viruses can be stabilized under theconditions set forth; for example, Coxsackie viruses, ECHG viruses andReoviruses.

In our copending application, Serial No. 186,618, filed April 11, 1962,we described processes and compositions of matter having to do withtissue culture mediums and cell cultures suitable for the production andfor the safety testing of viral vaccines. Accordingly, these are notdescribed and claimed in this application.

The present invention is therefore well suited and adapted to carry outthe objects and has the advantages mentioned as Well as others inherenttherein.

While several presently-preferred examples of the invention have beengiven for the purpose of disclosure, no undue restrictions orlimitations are to be imposed by reason thereof but that the presentinvention is defined by the appended claims.

What is claimed is:

1. The process which comprises adding a concentrated aqueous solutioncontaining at least one member of the group consisting of magnesiumchloride, magnesium sulfate, calcium chloride, zinc chloride, manganesesulfate, cobalt chloride, aluminum chloride, aluminum sulfate, andmixtures thereof to an aqueous suspension containing at least one liveribonucleic acid-containing tissue-culture propagated enterovirus,attenuated to avinulence to man, in concentrations effective toinactivate adventitious tissue culture contaminants.

2. A composition of matter consisting of an aqueous suspensioncontaining at least one live ribonucleic acidcontaining tissue-culturepropagated enterovirus, attenuated to avirulence to man, prepared byadding to said suspension a concentrated aqueous solution containing atleast one member of the group consisting of magnesium chloride,magnesium sulfate, calcium chloride, zinc chloride, manganese sulfate,cobalt chloride, aluminum chloride, aluminum sulfate, and mixturesthereof.

References Cited in the file of this patent UNITED STATES PATENTS2,529,461 Schneiderw-ir-th Nov. 7, 1950 2,946,724 Valentine July 26,1960 2,966,443 Cox Dec. 27, 1960 3,035,980 Tint et a1 May 22, 19623,048,523 Underwood Aug. 7, 1962 3,096,249 Prigal July 2, 1963 OTHERREFERENCES Schaelfer et al.: Purification of Poliomyelitic Virus,Archives of Pathology, vol. 15, pp. 221-226, 1933.

Stanley: Handbuch Ver Virusforschung, 1938, vol. 1, pp. 449-451.

Smolens et al.: Science 122, pages 240-241, 1955.

Gard: World Health Organization Monograph Series, No. 26 (1955), pages230-235.

Fraenkel-Conrat: N.Y. Academy of Sciences, Special Publ. No. 5, pages219-227, 1957.

Padgett: J. of Experimental Med., November 1, 1958, pages 651-664.

Rhodes et al.: Textbook of Virology, 1958, pages 59- 60, pub. byWilliams and Wilkins, Balt., Md.

Soine et al.: Rogers Inorganic Pharmaceutical Chemistry', 7th Edition,1961, pub. by Lea and Febiger, Phila, Pa., pp. 463-464, 475, and637-638.

Wallis et al.: Stabilization of Poliovirus by Cations, Texas Rep. Biol.Med. :19, pp. 683-700, Fall 1961.

Wallis et al.: Cationic Inactivation of Vacuolating Virus (SV-40) inPoliovirus Suspensions, Tex-as Rep. Biol. Med. 19, pp. 701-705, Fall1961.

1. THE PROCESS WHICH COMPRISES ADDING A CONCENTRATED AQUEOUS SOLUTIONCONTAINING AT LEAST ONE MEMBER OF THE GROUP CONSISTING OF MAGNESIUMCHLORIDE, MAGNESIUM SULFATE, CALCIUM CHLORIDE, ZINC CHLORIDE, MANGANESESULFATE, COBALT CHLORIDE, ALUMINUM CHLORIDE, ALUMINUM SULFATE, ANDMIXTURES THEREOF TO AN AQUEOUS SUSPENSION CONTAINING AT LEAST ONE LIVERIBONUCLEIC ACID-CONTAINING TISSUE-CULTURE PROPAGATED ENTEROVIRUS,ATTENUATED TO AVIRULENCE TO MAN, IN CONCENTRATION EFFECTIVE TOINACTIVATE ADVENTITIOUS TISSUE CULTURE CONTAMINANTS.